1. Field of the Invention
This invention relates generally to a process for the separation of acid protease from whole glucoamylase product, and more specifically to separating Aspergillus niger acid protease from a glucoamylase product by contacting the glucoamylase/protease mixture with an anionic exchange medium.
2. Description of the Related Art
The culture broth resulting from the fermentation of certain fungi, particularly Aspergillus niger, contains a mixture of various enzymes having differing activities, not necessarily compatible with one another. Although the addition of Aspergillus niger acid protease enzymes to a steeping solution or alcohol fermentation improves bacterial growth by increasing the availability of peptides and amino acids, this growth benefit is countered by the deleterious effects acid protease has to the glucoamylase present in the culture broths. Glucoamylases that are high in acid protease can lose activity rapidly, especially when stored at elevated temperatures, greatly decreasing the storage stability of glucoamylase products.
The instability of glucoamylase appears to be the result of hydrolysis of the glucoamylase protein by acid protease. Glucoamylase instability is still present when the fermentation broth is converted to whole glucoamylase product, which despite its name is a mixture of glucoamylase and acid protease, most of the remaining enzymes having been removed.
Accordingly, it is highly desirable to separate the glucoamylase enzymes from the destabilizing acid protease enzymes present in a fungal enzyme preparation. Reduction or elimination of acid protease from whole glucoamylase product would thus result in a more stable glucoamylase product.
Various techniques are known to purify amylases having various impurities, including acid proteases. For example, U.S. Pat. No. 2,121,459 to Waldschmidt-Leitz describes a process of increasing the ratio of amylolytic to proteolytic enzymes by adsorbing the proteases on bauxite.
U.S. Pat. No. 3,416,997 to Barton describes a process of purifying fungal alpha amylase from various fermentation products of fungi, particularly Aspergillus oryzae, utilizing an anionic ion-exchange material. The impurities removed by the exchanger include acid protease. Similarly, U.S. Pat. Nos. 3,335,066 and 3,345,268 to Corman describe processes for purifying an amyloglucosidase-containing fungal enzyme preparation of transglucosidase using an ion exchange resin. All of these references however, fail to disclose a means for the separation and collection of protease fungal enzymes.
U.S. Pat. No. 4,532,213 to Shetty et al. describes a method for recovering acid protease from fungal mycelia. The protease is produced during fermentation by growing the fungus in an aqueous medium, producing both glucoamylase and acid fungal protease which has become associated with the fungal mycelium. After separating the protease from the fungal mycelium, the protease is recovered by treating the mycelium/protease with a solution having a pH of about 5.0 to 6.5, and the mycelium is then separated out.
The above references, although generally recognizing the desirability of separating protease from amylase, fail to provide a technique for recovering both glucoamylase and acid protease using an anion exchange medium.
It is one object of the present invention to provide a simple and effective process for separating acid protease from glucoamylase.
Another object of the present invention is to provide simple and effective processes of separating and purifying the proteolytic protease enzyme of Aspergillus niger from glucoamylase without any appreciable loss in substance and activity of either.
Still another object of the present invention is to provide a more stable glucoamylase product.